In this R15 application, we seek to develop an innovative organoid development platform from human induced pluripotent stem cells (iPSCs) by unlocking oxygen signaling pathways that affect islet organogenesis and morphogenesis within three-dimensional (3D) scaffolds. Lab-regenerated islets can serve as disease models for study of diabetes-related pathophysiology and precision medicine, for drug screening and validation, and for islet transplantation. Despite intense efforts made to produce biologically functional islets, it is still an extremely challenging task. Efforts so far have focused primarily on identifying signal molecules and culture conditions to direct iPSC progression into pancreatic ? cells. We, on the other hand, are focusing on 3D scaffold culture, which is critical to islet development from iPSCs, as a carefully designed scaffold can offer the physiochemical niches that mimic microenvironments in the body. This was demonstrated in our earlier work. Nevertheless, oxygen supply becomes a limiting factor in a scaffold culture, due mainly to the lack of vascular structures inside the scaffold to facilitate mass and gas transfer. While vascularization is an ultimate solution, it is challenging to create. In addition, growing evidence suggests that normoxia or hyperoxia is essential for islet development and ? cell viability and metabolic activities. In our earlier study, we discovered that a scaffold?s cellular hypoxic condition can be eliminated by culturing ? cells with oxygenators that release and supply oxygen directly to the cells within the scaffolds. We developed, in another study, a technology that enables us to generate human islet organoids from iPSCs. The islet tissues generated consist of four different types of pancreatic endocrine cells: ?, ?, ?, and pancreatic polypeptide cells. They secrete insulin in response to glucose challenges. Based on these and other studies, we hypothesized that an in situ oxygen supply in oxygenation-enabled 3D scaffolds promotes organogenesis and morphogenesis of islet organoids from iPSCs. The development of an oxygenation-enabled scaffold culture platform for islet organoid formation from iPSCs has not yet been explored. To address this unmet need, we will characterize the effect of oxygen level on the structure and function of islets developed from iPSCs using an in situ oxygenated cell-laden scaffold culture platform. Two aims are proposed: 1) Developing a translational oxygenation-enabled 3D stem cell differentiation platform for islet development, and 2) Interrogating oxygen signal regulated islet organogenesis and morphogenesis within oxygenated scaffolds. Several human iPSC lines will be tested to validate the robustness of the technology developed. The proposed studies will shed light on the regulation of islet development by oxygen signaling pathways. They will also help design a better 3D organoid development system for generating biocomparable islets from iPSCs. Furthermore, knowledge gained through this study will contribute to the biofabrication of islets and other clinically relevant tissues from iPSCs.